JP2010539971A - Primer for PCR amplification containing an abasic part in the sequence - Google Patents

Abstract

The present invention relates to a PCR amplification primer containing an basic base in a sequence, and a PCR amplification method using the primer, and more specifically, a pair of primers containing an basic part at a polymorphic site of a gene, PCR including a PCR amplification primer that can amplify different templates having mutation sites in common, and a step of performing PCR amplification on the mixture after mixing the nucleic acid template with the PCR amplification composition containing the primer The present invention relates to an amplification method. The PCR amplification primer of the present invention has the advantage that different templates having mutation sites can be amplified in common by including an abasic portion having no specific coding information in the base sequence. There is.
[Selection] Figure 9

Description

  The present invention relates to a primer and a PCR amplification method using the same, more specifically, having a basic part in a primer complementary to a mutation or polymorphism site of a template DNA, The present invention relates to a primer capable of commonly amplifying different templates and a PCR amplification method using the same.

  The most commonly used nucleic acid amplification method, known as the polymerase chain reaction (hereinafter referred to as “PCR”), involves denaturation of double-stranded DNA, annealing of oligonucleotide primers to a DNA template, and primer extension by DNA polymerase. Includes repeated cycle processes (Mullis et al., US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al, 1985). The oligonucleotide primers used for PCR are designed to be annealed to the opposite strand of the DNA, and the primer's DNA polymerase extension product acts as a template strand for the other primers. The PCR amplification process results in an exponential increase of the DNA sequence, and the length of the amplified DNA sequence is determined by the 5′-end of the oligonucleotide primer.

  Since the success in nucleic acid amplification, in particular PCR amplification, depends on the specificity of the primers to anneal only to their target sequence, it is important to optimize the molecular interaction. Whether the primer anneals only to its perfect complement or even a sequence having one or more mismatched nucleotide sequences is determined by the annealing temperature. In general, the higher the annealing temperature, the higher the possibility of specific annealing of the primer with respect to the perfect match template, and thus the possibility that only the target sequence will be amplified. A lower annealing temperature results in tolerance to more mismatches between the template and the primer, eventually increasing amplification for non-target sequences. By adjusting the annealing temperature, the pairing specificity between the template and the primer can be changed. For example, if other products occur in the control group where only one primer is present, such a result indicates that the single primer anneals to one or more sites in the template. In such a case, it is preferable to increase the annealing temperature. Considering the above effect of the annealing temperature on the primer's annealing specificity, the primer annealing can be adjusted according to the annealing temperature, so that it is possible to improve the primer's annealing specificity regardless of the primer design. It turns out that there is a strong demand.

  Primer parameters such as primer length, GC amount and PCR product length as well as annealing temperature must be considered for primer annealing specificity. If primers that satisfy the above parameters are used, primer annealing will be specified and target DNA amplification will significantly improve primer annealing specificity, resulting in background problems and non-specific product problems introduced by the primers. Is resolved. Well-designed primers not only solve non-specific annealing and background problems, but also allow RNA-PCR to distinguish between cDNA or genomic templates.

  In many cases, the primer sequence does not seek to be a perfect complement to the template sequence. The part of the primer that must be perfectly matched with the template is the 3'-end because this end is extended by a DNA polymerase, which guarantees the annealing specificity for the correct target sequence. The end is most important. The 5'-end of the primer is not critical for determining the specificity of annealing to the target sequence and can be modified to carry additional sequences that are non-complementary to the template, such as restriction enzyme positions and promoter sequences.

  Molecular diagnostic testing or nucleic acid diagnostic testing is the fastest growing field in the in vitro diagnostics market, with an average annual growth rate of approximately 20% in the global market. Molecular diagnostic testing has shown an 8% share in the in vitro diagnostic test market of about 30 trillion won in 2003, but it accounted for more than 13% in 2008, with sales exceeding 5 trillion won, an average annual The growth rate is expected to be around 19%. Molecular diagnostic testing is a method for testing disease-causing viruses, bacteria, fungi, etc. by PCR, and infecting pathogens using oligonucleotides (primers, probes) that are complementary to the base sequence of a specific pathogen gene. Check for presence. The pathogen infection detection method using PCR is used as an application method for determining positive and negative by specifically detecting only the gene using only the primer specific to the template gene. In comparison with the antigen-antibody detection method using ELISA (enzyme-linked immuno-sorbent assay), there is an advantage that high sensitivity and economy can be secured.

  Multiplex PCR is another variation of PCR that can simultaneously amplify one or more target sequences using one or more primer pairs in the same reaction. Since its first introduction in 1988 (Chamberlain et al., Deletion Screening of the Duchenne muscular dystrophy locus via multiplex DNA Amplificationm Nucleic Acids Res., 16, 11141-11156, 1988), this method has been used to analyze gene deletions ( Anonymous, Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction, A multicenter study.JAMA 267, 2609-2615, 1992; Henegariu, et al., Rapid screening of the Y chromosome in idiopathic sterile men, diagnostic for deletions in AZF, a genetic Y factor expressed during spermatogenesis, Andrologia, 26, 97-106, 1994), mutation and gene polymorphism analysis (Shuber et al., Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis, Hum. Mol. Geenet., 2, 153-158, 1993; Mutirangura et al., Mutiplex PCR of three dinucleotide repeats in the Prader-Willi / Angelman critical region (15q11-q13): molecular diagnosis and mechanism of uniparental disomy, Hum Mol. Genet., 2, 143-151, 1993), quantitative analysis (Zimme rmann et al., Quantitative multiplex competitive PCR of HIV-1 DNA in a single reaction tube, BioTechniques 21, 480-484, 1996) and RNA detection (Zou et al., Identification of new influenza B virus variants by multiplex reverse transcription- It has been successfully used in various fields of DNA analysis, including PCR and the heteroduplex mobility assay, J. Clin. Microbiol., 36, 1544-1548, 1998). In the field of infectious diseases, this technology is valued as a valuable method for the identification of viruses, bacteria, fungi and / or parasites.

  However, the results obtained from multiplex PCR are often complicated due to artifacts in the amplification process. Such false results include “false positive” results due to reaction failure, and “false positive” results such as amplification of false products, said false positive results being associated with the original cognitive sequence, Apparently occurs because the primer anneals to a different sequence. In multiplex PCR, the primer must be designed so that its hybridization kinetics are similar to those of other primers used in the same multiplex reaction. Although the annealing temperature and primer concentration can be calculated to some extent, generally conditions etc. must be determined empirically for each multiplex reaction. As the number of primer pairs increases, the possibility of non-specific priming increases, so the conditions and the like must be changed each time each primer pair is added. In addition, artifacts resulting from competition for reactants (e.g. primer depletion) are further increased in multiplex PCR, and the difference in yield of unequally amplified products becomes even greater as the cycle is advanced Because. Therefore, optimizing the reaction conditions for multiplex PCR is a work that requires a lot of human power and time. Since different multiplex PCRs have unique reaction conditions, the development of new diagnostic test methods is expensive.

  Single nucleotide polymorphism (hereinafter referred to as “SNP”) is a single nucleotide pair of base mutations representing individual deviations among the base sequences of chromosomes of race, age or gender. This refers to the case of differences, and is the most common DNA base sequence polymorphism. About 1 mutation per 1,000 genes appears in human genes, but even if the disease is the same, it is thought that all of the individual deviations are due to differences in SNPs. SNP analysis methods developed to date include SSCP (Single Strand Conformation Polymorphism), PCR-RFLP (Restriction Fragment Length Polymorphism), Allele-specific PCR (AS-PCR), and Tm-shift Genotype using GC-tail primers. Typing (genotyping), DASH (Dynamic allele-specific hybridization), Taqman (5'-exonuclease assay), Molecular Beacons, OLA (Oligonucleotide ligase assay), Pyrosequencing, single base extension, There are Fluorescence Polarization and MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight), etc., and these analysis methods are based on single oligonucleotides and probe oligonucleotides labeled with fluorescence for each template. Perform comparative analysis for base mutations.

  On the other hand, when the gene desired to be detected exists in the form of a large number of polymorphic genes including mutation positions in the sequence, a polymorphism is used to complement the mutation position for accurate gene amplification. After making it as a primer according to the type of sex, you must experiment. For this purpose, it is necessary to know all the polymorphic sequences for a specific gene. Therefore, if there is a polymorphic gene whose sequence is unknown, it is impossible to detect all desired genes. There is. Therefore, there is a demand for the development of a simple method capable of amplifying these mutation sites in common using a pair of primers in a specific base sequence having a restricted mutation or polymorphism.

  The present invention has been devised in order to improve the problems in the conventional PCR method as described above, and is a PCR in which different polymorphic sites of a gene to be amplified are replaced with an basic portion. It is an object of the present invention to provide an amplification primer and a PCR method that can amplify nucleic acids of different templates having mutation or polymorphic sites in the base sequence in common using the primer.

  In order to achieve the above object, the present invention provides a primer for PCR amplification containing an abasic portion in the sequence.

  In the present specification, “basic portion” refers to a nucleoside that does not have specific base coding information, and since there is no coding information, the base of the corresponding template DNA is adenine, guanine, cytosine, or thymine. Any of them may be used. The basic portion is preferably located at a position corresponding to a mutation site and / or a polymorphism site in the primer of the present invention. The basic portion includes dspacer (dSpacer, 5′-O-dimethoxytrityl-1 ′, 2′-dideoxyribose-3 ′-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), methoxy Deespacer (1'-OMe-dSpacer, 5'-O-dimethoxytrityl-1'-methoxy-2'-dideoxyribose-3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), PC PC Spacer Phosphoramidite, [4- (4,4'-Dimethoxytrityloxy) butyramidomethyl) -1- (2-nitrophenyl) -ethyl] -2-cyanoethyl- (N, N-diisopropyl) -phosphoramidite), Earl Spacer RSpacer CE Phosphoramidite (5'-O-Dimethoxytrityl-1'-Deoxyribose-2'-O-Triisopropylsilyloxymethyl-3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) Spacer C12 CE Phosphoramidite, 12- (4,4'-Dimethoxytrityloxy) -dodecyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), spacer phosphoramidite 18 (Spacer Phosphoramidite 18, 18-O-Dimethoxytritylhe xaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite 9,9-O-Dimethoxytrityl-triethyleneglycol, 1-[(2-cyanoethyl)-( N, N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite C3,3- (4,4'-Dimethoxytrityloxy) propyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)] Primer for PCR amplification including an abasic portion synthesized starting from -phosphoramidite). However, it is not necessarily limited to this. Even if another phosphoramidite is used with a slight structural change, if the basic part in the gene (primer) synthesized and used is the same structure, the present invention is an amplification primer containing the basic part. It belongs to the category of the present invention.

  By including an basic part in the primer sequence as described above, it is possible to amplify all templates having different polymorphic sites in the same gene into one primer pair. The number of basic portions in the primer is not particularly limited, but is preferably in the range of 1 to 10 including the 3 ′ end, the internal sequence and the 5 ′ end, and 1 to 3 More preferably, it is the range. If the number of basic parts exceeds 10, the Tm value of the primer becomes remarkably low and annealing to the template DNA base sequence cannot be performed well, which may cause a serious problem that PCR reaction is not performed. This makes it impossible to achieve the object of the present invention to amplify different templates having mutation sites in common.

  Furthermore, the present invention provides a PCR amplification composition comprising a reaction buffer solution, four types of dNTPs, a DNA polymerase, a probe, and the PCR amplification primer.

As the reaction buffer solution, a normal PCR amplification buffer solution containing components such as Tris-HCl, KCl, (NH ₄ ) ₂ SO ₄ , MgSO ₄ , and MgCl ₂ can be appropriately modified and used. The four types of dNTPs mean dATP, dTTP, dGTP and dCTP, and the DNA polymerase that can be used is not limited to a specific enzyme. In a preferred embodiment of the present invention, PCR amplification was performed using Taq DNA polymerase, Klen Taq DNA polymerase, Pfu DNA polymerase. Furthermore, the primer for PCR amplification can be used at a concentration in the range of 1 pmole to 50 pmole based on a 20 uL PCR reaction solution, and it is obvious that an appropriate primer concentration can be easily determined by those skilled in the art.

  The composition for PCR amplification of the present invention may further contain a dye and / or a stabilizer for the purpose of experimentation, prevention of contamination by PCR reaction products, stabilization of DNA polymerase and dNTP and improvement of reactivity. it can. At this time, one or more of bromophenol blue, xylene cyanole, bromocresol red, or cresol red can be used as the dye, and the stabilizer One or more of gelatin, bovine serum albumin (BSA), Thesit, PEG-8000 or polyhydric alcohol (Polyol) can be used, but dyes and stabilizers are always limited to these compounds It is not done. The content of the dye in the composition is in the range of 0.0001 to 0.01% by weight with respect to the final composition, preferably 0.001 to 0.005% by weight, and more preferably 0.001 to 0.003% by weight. Furthermore, the stabilizer in the composition has a concentration in the final composition in the range of 2 to 1,000 mM, preferably in the range of 100 to 500 mM, and more preferably in the range of 100 to 300 mM.

  Furthermore, the present invention provides a PCR amplification method using the PCR amplification primer containing an abasic portion in the sequence.

  The method includes the steps of mixing a template DNA with a PCR amplification composition containing the PCR amplification primer containing an basic portion in the sequence, and performing PCR amplification on the mixture.

  The PCR amplification method of the present invention can be preferably used for the purpose of commonly amplifying different nucleic acid templates having mutation sites and / or polymorphic sites in the base sequence, but is not limited thereto. It is not a thing. In addition, the primer pair used for PCR amplification may be used for all primers including the basic portion of the present invention, and only one of them may be used for the primer including the basic portion of the present invention. One of them may use a normal primer that does not contain an abasic portion.

  The PCR amplification is performed by repeating the denaturation, annealing, and extension steps, and the principle of such PCR amplification is obvious to those skilled in the art. The denaturation, annealing, and extension temperatures are preferably 85 to 95 ° C for 1 to 60 seconds, 40 to 70 ° C for 1 to 60 seconds, and 50 to 75 ° C for 1 to 60 seconds, respectively. Can be adjusted accordingly. For example, according to a preferred embodiment of the present invention, when one basic part is replaced inside the primer, the melting temperature (hereinafter referred to as “Tm”) is lowered by about 8 ° C., and the basic at the 3 ′ end is reduced. When one part is replaced, Tm is lowered by about 1.5 ° C. On the other hand, in order to compensate for the lower Tm in the basic portion, it is possible to adjust the PCR reactivity by adding bases complementary to the template at both ends of the primer. For example, when a complementary base is added to the 3 ′ end, Tm increases by about 0.5 ° C. every time two bases are added. In addition, when 1, 2, or 3 basic parts are replaced in the primer and 5 bases are added at the same time to the 5 'end, the Tm is about 4 ° C, about 10 ° C, and about 18 ° C, respectively. Becomes lower. Such a change in Tm temperature may vary somewhat depending on the type of DNA polymerase used, the length of the primer, the type of base in the primer, the reaction conditions, and the like. When designing a primer containing a mutation site inside based on the degree of Tm change as described above, it becomes possible to establish primer production conditions that give temperature equivalence, and introduce an abasic part as appropriate. By increasing the length of the primer, the specificity of the PCR reaction can be controlled, so that different templates having mutation sites in the base sequence can be amplified in common with only a pair of primers. become.

  According to one embodiment of the present invention (Examples 1 and 2), as a result of PCR amplification for each DNA polymerase using human genomic DNA as a template and using a primer replaced with the basic portion, 1 in the case of Taq DNA polymerase. A Tm reduction of about 3-6 ° C is observed only with base substitution, a Tm reduction of about 6 ° C is observed only with 1 base substitution with Pfu DNA polymerase, and a single base with Klen Taq DNA polymerase. Although a Tm decrease of about 1 to 2 ° C. was observed in the replacement, it was confirmed that there was no problem in the PCR reaction. Therefore, it can be seen that the primer including the basic portion is applicable to various types of DNA polymerases. In addition, appropriate and optimal basic primer conditions for each DNA polymerase can be set, and based on these selection conditions etc., establish primer production conditions that give temperature equivalence when designing primers that include mutations for each DNA polymerase. Will be able to.

  According to another example of the present invention (Example 4), in the case of 1 base substitution, if 1 base is replaced with the basic part at the 3 ′ end of the primer, Tm is about 1.5 ° C. compared to the control primer pair. If the base sequence is replaced with 1 base, it decreases by about 8 ° C. Furthermore, if 5 bases complementary to the template are added at the 3 ′ end by replacing the 1 base, the Tm decreases by about 4 ° C. compared to the control primer pair, and the 2 bases are replaced at the 3 ′ end. If 5 bases complementary to the template are added, the temperature decreases by about 10 ° C. If 3 bases are replaced and 5 bases complementary to the template are added to the 3 ′ end, the temperature decreases by about 18 ° C. If you replace 1 base and add 1 base complementary to the template at the 3 'end, it will decrease by about 5 ° C. If you replace 1 base and add 3 base complementary to the template at the 3' end The temperature decreases by about 4.5 ° C. If 5 bases complementary to the template are added to the 3 'end by replacing the 1 base, the temperature decreases by about 4 ° C. The results are summarized in Table 1.

  Interpreting the above results, the Tm value at about 6.5 ° C. is reduced when replacing one base at both ends compared to when replacing one base at both ends. When 5 bases are added to the 3 'end at the time of 1 base substitution, 2 base substitutions, and 3 base substitutions, about 6 ° C to 8 ° C Tm value as the number of bases to be replaced increases The Tm value is about 0.5 ° C as 2 bases are added at the 3 'end, such as 1 base addition at the 3' end, 3 base additions, and 5 base additions for the inherent 1 base substitution. Increase by increments.

  By using the above results, it is possible to establish primer production conditions that give temperature equivalence at the time of primer design with respect to the primer position, and base sequences that minimize a significant difference in Tm value that can be generated by introducing an abasic part. Can be produced, thereby increasing the specificity of the PCR reaction.

  As discussed above, the primers for PCR amplification of the present invention containing the basic portion in the sequence can be obtained from each other having a mutation site in the base sequence by including the basic portion in the primer base sequence. Different templates can be amplified in common using a pair of primers.

2 is an electrophoretogram showing the results of amplification by Taq. DNA polymerase using a nucleic acid amplification method (polymerase chain reaction, hereinafter referred to as “PCR”) using a p55 / p53 primer pair containing an basic portion. It is an electrophoresis photograph showing the result of PCR amplification with Klen Taq. DNA polymerase using a p55 / p53 primer pair containing an basic portion. It is an electrophoresis photograph showing the result of PCR amplification with Taq. DNA polymerase using p63 / p55 primer pair containing the basic portion. It is the electrophoresis photograph which shows the result of PCR amplification by Pfu DNA polymerase using the p63 / p55 primer pair containing an abasic part. For the control group primer pair and the control group primer pair, a primer pair in which the basic part is replaced by 1 base at the 3 ′ end, a primer pair in which one base is replaced in the internal base sequence, and a base pair in which the internal base sequence is replaced by 3 bases 'Electrophoresis photographs showing the results of PCR amplification with Klen Taq. DNA polymerase using primer pairs added with 5 bases complementary to the ends. Compared to the control group primer pair, the basic part is replaced with 3 bases in the internal base sequence and 5 bases complementary to the 3 'end is added, and the base pair is replaced with 1 base in the internal base sequence and complementary to the 3' end A primer pair with one additional base, a primer pair with three bases replaced by one base to the 3 'end and a base pair with three bases added complementary to the 3' end It is an electrophoresis photograph showing the results of PCR amplification with Klen Taq. DNA polymerase using each primer pair with 5 bases added. It is a melting curve result graph which created the primer pair from which the replacement position of an basic part differs using the real-time gene amplification apparatus. It is a melting curve result graph which created the primer pair from which the number of internal base sequence substitutions of an basic part differs using the real-time gene amplification apparatus. It is a melting curve result graph which created the primer pair from which the basic part replaced 1 base inherently, and was added to 3 'terminal with a different number of bases using the real-time gene amplifier. In FIG. 7 to FIG. 9, the purple line is a line representing a decrease change in the fluorescence value on the y-axis accompanying a change in temperature increase on the x-axis in each figure. It is a figure which shows the position where the basic part was replaced by 1 base with respect to the primer with respect to the polymorphic site | part of the S gene of hepatitis B virus. Shows the results of real-time PCR using a primer (dS) in which one base was replaced with a deespacer, a primer (Me-dS) that was replaced with a methoxydeespacer, or a primer (Reg) consisting of normal bases and a probe. In the graph, each line or the like is a line representing a change in fluorescence value with an increase in the number of PCR amplification cycles for three types of standard samples having different concentrations. Shows the results of real-time PCR using a primer (dS) in which one base was replaced with a deespacer, a primer (Me-dS) that was replaced with a methoxydeespacer, or a primer (Reg) consisting of normal bases and a probe. In the graph, each line or the like is a line representing a change in fluorescence value with an increase in the number of PCR amplification cycles for three types of standard samples having different concentrations.

  Hereinafter, the present invention will be described in more detail through examples.

  However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Verification of the effectiveness of PCR amplification using p55 / p53 primers containing the basic portion
A basic product based on p55 / p53 primer pair using Taq DNA polymerase (Bionia, hereinafter abbreviated as “Taq”) and Klen Taq. DNA polymerase (Bionia, abbreviated as “Klen Taq”). The nucleic acid amplification reactivity and specificity by the primer were confirmed. Therefore, human genomic DNA was used as the template DNA, and the following three primer pairs were used as the primer pair: p55 primer (base sequence: 5 '-CTC TTC CTG CAG TAC TCC CCT GC-3', SEQ ID NO: 1) and p53 primer (base sequence: 5'-GCC CCA GCT CAC CAT CGC TA-3 ', SEQ ID NO: 2), each 15 pmole per 20 μl reaction Used one by one. In the first sample group primer pair, “N” (D Spacer, Glen Research, Cat. No. 10-1914-xx) is used instead of “C” which is the 10th base sequence from the 5 ′ end of the p55 primer. No2-1F (SEQ ID NO: 3) replaced with, and No2-1R (SEQ ID NO: 4) replaced with `` N '' (deespacer) instead of `` T '' which is the ninth base sequence from the 5 ′ end of the p53 primer ) Was used at 15 pmole per 20 μl reaction. In the second sample group primer pair, No2-2F (SEQ ID NO: 5) replaced with `` NN '' (D spacer) instead of CA which is the 10th and 11th base sequences from the 5 ′ end of the p55 primer, No2-2R (SEQ ID NO: 6) replaced with “NN” (D spacer) instead of TC, which is the 9th and 10th nucleotide sequences from the 5 ′ end of the p53 primer, was used at 15 pmole per 20 μl reaction.

For the enzyme for PCR reaction, 1 U (U, unit) Taq DNA polymerase per 20 μl reaction or 0.4 U (U, unit) Klen Taq DNA polymerase per 20 μl reaction (Bionia, hereinafter abbreviated as “Klen Taq”) ) Was used. For other reaction additives, add 2 μl of dNTP mixture (2.5 mM of each dATP, dCTP, dGTP and dTTP mixture) per 20 μl reaction, 100 mM Tris-HCl, 400 mM as a 10 × reaction buffer for Taq and Klen Taq enzymes. ₂ μl of KCl, 15 mM MgCl ₂ , pH 9.0 was added per 20 μl reaction.

  The reaction conditions for PCR amplification are as follows. In the case of Taq, proceed with a pre-denaturation step at 94 ° C for 5 minutes, followed by a denaturation step at 94 ° C for 30 seconds, an annealing step at 45 ° C to 59 ° C for 50 seconds, and an extension step at 72 ° C for 50 seconds. The reaction was performed in 38 reaction cycles in total, and the final extension step was advanced at 72 ° C. for 5 minutes. Furthermore, in the case of Klen Taq, a non-specific reaction was induced at 37 ° C for 5 minutes, followed by a pre-denaturation step at 94 ° C for 5 minutes, followed by a denaturation step at 94 ° C for 30 seconds and gradually from 45 ° C to 59 ° C. Under the conditions, an annealing step for 50 seconds and an extension step for 50 seconds at 72 ° C. were performed for a total of 38 reaction cycles, followed by a final extension step at 72 ° C. for 5 minutes. The amplification result for the PCR reaction was confirmed by electrophoresis using 0.5x TBE buffer (Tris base, Boric Acid and 0.5M EDTA, pH 8.0) containing 2.0% agarose.

  FIG. 1 shows agarose gel electrophoresis results for PCR reaction products using a control group primer pair for Taq, a first sample group primer pair, and a second sample group primer pair. Lane 1 to Lane 10, Lane 11 to Lane 20 and Lane 21 to Lane 30 show cases where a control group primer pair, a first sample group primer pair, and a second sample group primer pair were used, respectively. The reaction results when the annealing temperatures are 45.1 ° C, 45.3 ° C, 46.3 ° C, 47.7 ° C, 49.4 ° C, 51.4 ° C, 53.3 ° C, 55.3 ° C, 57.6 ° C and 59.0 ° C, respectively, M is 100 to 2,000 bp. It is a DNA size marker (Bionia) that can confirm the range. The DNA size marker includes a total of 13 DNA double helix DNA pieces, specifically, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1,000 bp, 1,200 bp. Including DNA double helix pieces of 1,600 bp and 2,000 bp size. In the case of the control group, two types of bands appear strongly, the upper band represents the target band, the lower band represents the primer dimer band result, and the non-specific reaction is seen from above the target band. While the band is confirmed.

  From the results shown in FIG. 1, the non-specific reaction and primer dimer appearance decreased under the conditions of the first sample group primer pair and the second primer pair, particularly in the case of the first primer pair, the non-specific reaction and the primer dimer than the second primer pair. It was confirmed that the aspect decreased more. Furthermore, a high PCR reaction product was confirmed.

  On the other hand, when comparing the Tm values, in the case of the control group (lanes 1 to 10), the annealing temperature was 45.1 ° C, 45.3 ° C, 46.3 ° C, 47.7 ° C, 49.4 ° C, 51.4 ° C, 53.3 ° C, 55.3 ° C, 57.6 When the temperature was 5 ° C. and 59.0 ° C., the template was all amplified, but the first sample group primer pair (lanes 11 to 20) was amplified only until the 18th lane, ie, the annealing temperature was 55.3 ° C. Therefore, ΔTm = 59.0 ° C.-55.3 ° C. = 3.7 ° C. In the second sample group primer pair (lanes 21 to 30), amplification was performed only until the 26th lane, that is, when the annealing temperature was 51.4 ° C. Therefore, ΔTm = 59.0 ° C.-51.4 ° C. = 7.6 ° C.

  FIG. 2 shows agarose gel electrophoresis results for PCR reaction products using a control group primer pair for Klen Taq, a first sample group primer pair, and a second sample group primer pair. The explanation for lanes 1 to 30 and M is the same as in the first embodiment. From the results of FIG. 2, it was confirmed that the non-specific reaction and the primer dimer appearance decreased under the conditions of the first sample group primer pair and the second sample group primer pair.

  On the other hand, if Tm values are compared by the same method of FIG. 1, ΔTm of the first sample group primer pair and the second sample group primer pair is 1.4 ° C. and 5.7 ° C., respectively.

  From the above results, it was confirmed that the nonspecific reaction was reduced in the Taq enzyme and the Klen Taq enzyme in the case of the 1-base substitution of the first sample group primer, and also in the case of the 2-base substitution of the second sample group primer. It was confirmed that non-specific reaction decreased with Taq enzyme and Klen Taq enzyme. That is, by introducing the basic part, it can be seen that the non-specific reaction is not increased but decreased, so the primer of the present invention in which the basic part that is not complementary to the template is introduced is a reaction-specific primer. It was verified that the sex was maintained. It was confirmed that the annealing temperature for the PCR reaction decreased slightly in all the applied DNA polymerases under the condition that the specific base was replaced with an abasic part (deespacer) on the sample group primer base sequence.

Example 2. Verification of the effectiveness of PCR amplification using p63 / p55 primers containing the basic portion
Taq and Pfu DNA polymerase (Bionia, abbreviated as “Pfu” hereinafter) were used to confirm the nucleic acid amplification reactivity and specificity for the basic primer produced based on the p63 / p55 primer pair. Therefore, human genomic DNA was used as the template DNA, and the following three primer pairs were used as the primer pair: p55 primer (base sequence: 5) having an amplification size of 447 bp as the control group primer pair '-CTC TTC CTG CAG TAC TCC CCT GC-3', SEQ ID NO: 1) and p63 primer (base sequence: 5'-GGC CAC TGA CAA CCA CCC TTA CC-3 ', SEQ ID NO: 7) per 20 μl reaction 15pmole was used. In the first sample group primer pair, No3-1R (SEQ ID NO: replaced with “N” (deespacer) instead of “C” which is the 10th base sequence from the 5 ′ end of the No2-1F and p63 primers. 8) was used at 15 pmole per 20 μl reaction. In the second sample group primer pair, No3-2R replaced with “NN” (D spacer) instead of “CA” which is the 10th and 11th base sequences from the 5 ′ end of the No2-2F and p63 primers. (SEQ ID NO: 9) was used at 15 pmole per 20 μl reaction.

As the enzyme for the PCR reaction, 1 U Taq or Pfu was used per 20 μl reaction. For other reaction additives, dNTP mixture (2.5 mM of each dATP, dCTP, dGTP, and dTTP mixture) was added at 2 μl per 20 μl reaction, and 100 mM Tris-HCl, 400 mM KCl, 10 × reaction buffer for Taq enzyme, Add 20 μl of 15 mM MgCl ₂ , pH 9.0 per reaction, 2 μl per reaction, 10x reaction buffer for Pfu enzyme is 200 mM Tris-HCl, 100 mM KCl, 100 mM (NH ₄ ) ₂ SO ₄ , 20 mM MgSO ₄ , 1% Triton X-100 1 mg / ml acetylated BSA, pH 8.8, was added in 2 μl per 20 μl reaction.

  The reaction conditions for the PCR reaction are as follows. Induce an artificial non-specific reaction at 37 ° C for 5 minutes, proceed with a pre-denaturation step at 94 ° C for 5 minutes, followed by a denaturation step at 94 ° C for 30 seconds and an annealing step gradually at 45 ° C to 59 ° C for 50 seconds In addition, the extension step was performed at 72 ° C. for 50 seconds for one reaction cycle for a total of 38 reaction cycles, and then the final extension step was advanced at 72 ° C. for 5 minutes. The amplification result for the PCR reaction was confirmed by electrophoresis using 0.5 × TBE buffer containing 2.0% agarose.

  FIG. 3 shows agarose gel electrophoresis results for PCR reaction products for a control group primer pair for Taq, a first sample group primer pair, and a second sample group primer pair. The explanation for lanes 1 to 30 and M is the same as in the first embodiment. From the results of FIG. 3, the non-specific reaction and the primer dimer aspect decreased under the conditions of the first sample group primer pair and the second sample group primer pair, and particularly good results were confirmed in the case of the first primer pair. On the other hand, ΔTm is 5.7 ° C. and 7.6 ° C., respectively.

  FIG. 4 is an agarose gel electrophoresis result for a PCR reaction product using a control group primer pair for Pfu, a first sample group primer pair, and a second sample group primer pair. The explanation for lanes 1 to 30 and M is the same as in the first embodiment. From the results of FIG. 4, the non-specific reaction and the primer dimer aspect decreased under the conditions of the first sample group primer pair and the second sample group primer pair, and in particular, the better result was confirmed with the second primer pair. On the other hand, ΔTm is 5.7 ° C. and 7.6 ° C., respectively.

  From the above results, in all cases where Taq and Pfu were used, the reactivity of the first primer pair and the second primer pair increased, and the non-specific reaction decreased. From this, it is confirmed that even basic primers are effective in PCR reactivity and specificity, and when a specific base is replaced with an abasic part (de-spacer), all applied DNA polymerases can be used for PCR reactions. It was confirmed that the annealing temperature slightly decreased.

Example 3. Verification of the effectiveness of DNA polymerase against basic primer production conditions Based on the control group primer pair, 1 base substitution at the 3 ′ end (sample 2), 1 base substitution at the internal base sequence (sample 3), internal base 2 base substitutions in the sequence and 5 bases complementary to the 3 'end (sample 4), 3 bases substitution in the internal base sequence and 5 bases complementary to the 3' end (sample 5), 1 base in the internal base sequence 1 base addition complementary to the 3 'end (sample 6), 1 base replacement to the internal base sequence, 3 bases complementary to the 3' end (sample 7), and 1 base replacement to the internal base sequence 3 ' Basic primers were prepared in 7 cases with 5 base additions (sample 8) complementary to the ends.

  Human template DNA was used as the template DNA, and 12 pmole to 40 pmole per 20 μl reaction was used as the primer pair. First, sample 1 is an F_AP_CONT primer (base sequence: 5'-CGT GTT TGT GCC TGT CCT GG-3 ', SEQ ID NO: 10) and an R_AP_CONT primer (base sequence: 5' CCG CTT CTT GTC CTG CTT GC-3 ', SEQ ID NO: 11) pair, sample 2 is F_AP_CONT_3-1N primer (base sequence: 5'-) as a primer pair in which 1 base at the 3' end is replaced with the control group primer CGT GTT TGT GCC TGT CCT GN-3 ', SEQ ID NO: 12) and R_AP_CONT_3-1N primer (base sequence: 5'-CCG CTT CTT GTC CTG CTT GN-3', SEQ ID NO: 13) pair, sample 3 is a control F_AP_CONT_I-1N primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GG-3 ', SEQ ID NO: 14) and R_AP_CONT_I-1N primer (base sequence: 5'-CCG CTT CTT GTN CTG CTT GC-3 ', SEQ ID NO: 15) pair was used, and in sample 4, the 3' end of the control group primer was replaced with 2 base nucleotide sequences. F_AP_CONT_I-2N_3 + 5M primer (base sequence: 5'-CGT GTT TNT GCN TGT CCT GGG AGA G-3 ', SEQ ID NO: 16) and R_AP_CONT_I-2N_3 + 5M primer (Base sequence: 5'-CCG CTT NTT GTN CTG CTT GCT TAC C-3 ', SEQ ID NO: 17) pair, sample 5 is the control group primer is replaced with 3 base nucleotide sequence, 3' terminal 5 base F_AP_CONT_I-3N_3 + 5M primer (base sequence: 5'-CGT GTT TNT GCN TGT NCT GGG AGA G-3 ', SEQ ID NO: 18) and R_AP_CONT_I-3N_3 + 5M primer (base sequence: 5'- CCG CTT NTT GTN CTG NTT GCT TAC C-3 ', SEQ ID NO: 19) pair, sample 6 is F_AP_CONT_I as a primer pair in which 1 base of the internal base sequence is replaced with the control group primer and 1 base at the 3' end is added -1N_3 + 1M primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG-3 ', SEQ ID NO: 20) and R_AP_CONT_1N_3 + 1M primer (base sequence: 5'-CCG CTT CTT GTN CTG CTT GCT-3' (SEQ ID NO: 21) pair, sample 7 was F_AP_CONT_I-1N_3 + 3M primer (base sequence: 5'-CGT) as a primer pair in which 1 base of the internal base sequence was replaced with the control group primer and 3 bases of 3 bases were added. Sample using GTT TGT GCN TGT CCT GGG AG-3 ', SEQ ID NO: 22) and R_AP_CONT_I-1N_3 + 3M primer (base sequence: 5'-CCG CTT CTT GTN CTG CTT GCT TA-3', SEQ ID NO: 23) 8 is an F_AP_CONT_I-1N_3 + 5M primer pair (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG AGA G- 3 ′, SEQ ID NO: 24) and R_AP_CONT_I-1N_3 + 5M primer (base sequence: 5′-CCG CTT CTT GTN CTG CTT GCT TAC C-3 ′, SEQ ID NO: 25) pair were used.

As the enzyme for the PCR reaction, 0.4 U (U, unit) of Klen Taq enzyme per 20 μl reaction was used. For other reaction additives, dNTP mixture (2.5 mM each dATP, dCTP, dGTP and dTTP mixture) was added in 2 μl per 20 μl reaction, and 100 mM Tris-HCl, 400 mM KCl as 10 × reaction buffer for Klen Taq enzyme. ₂ μl per 20 μl reaction of 15 mM MgCl ₂ , pH 9.0 was added.

  The reaction conditions for PCR amplification are as follows. Proceed with a pre-denaturation step at 94 ° C for 5 minutes, followed by a denaturation step at 94 ° C for 30 seconds and an annealing step gradually at 55 ° C to 70 ° C or 46 ° C to 61 ° C, and 50 ° C at 72 ° C. The reaction step was advanced 40 times in total, with an extension step of 1 second per reaction cycle, and then the final extension step was advanced at 72 ° C. for 5 minutes. The reaction amplification result for the PCR reaction was confirmed by electrophoresis using 0.5x TBE buffer containing 2.0% agarose.

  FIG. 5 and FIG. 6 are agarose gel electrophoresis results of PCR reaction products for the eight primer pairs for the Klen Taq enzyme, and the explanation for M is the same as in Example 1.

In FIG. 5, lane 1 to lane 8 show the reaction results when the annealing temperature is 59.4 ° C., 61.4 ° C., 63.3 ° C. , 65.3 ° C., 67.6 ° C., 69.0 ° C., 69.7 ° C., and 70.2 ° C. sequentially for the sample 1 primer pair. Lane 9 to Lane 16 are the reaction results when the annealing temperature is 55.5 ° C, 56.3 ° C, 57.1 ° C, 59.4 ° C, 61.4 ° C , 63.3 ° C, 65.3 ° C, and 67.6 ° C sequentially for the sample 2 primer pair. Lanes 17 to 24 are the reaction results when the annealing temperatures are sequentially 46.1 ° C, 46.5 ° C, 47.3 ° C, 48.7 ° C, 50.4 ° C, 52.4 ° C, 54.3 ° C, and 56.3 ° C with respect to the sample 3 primer pair, Lanes 25 to 32 show the reaction results when the annealing temperature is 48.7 ° C., 50.4 ° C., 52.4 ° C. , 54.3 ° C., 56.3 ° C., 58.6 ° C., 60 ° C. and 60.7 ° C. sequentially for the sample 4 primer pair. Is a 100 bp DNA size marker.

Furthermore, in FIG. 6, lanes 1 to 8 are reactions when the annealing temperatures are sequentially 46.1 ° C., 46.5 ° C., 47.3 ° C., 48.7 ° C., 50.4 ° C., 52.4 ° C., 54.3 ° C., and 56.3 ° C. for the sample 5 primer pair. The lanes 9 to 16 are the reaction results when the annealing temperature is 46.1 ° C., 46.5 ° C., 47.3 ° C., 48.7 ° C., 50.4 ° C., 52.4 ° C., 54.3 ° C., and 56.3 ° C. sequentially with respect to the sample 6 primer pair. Lanes 17 to 24 show the reaction results when the annealing temperature is 46.1 ° C, 46.5 ° C, 47.3 ° C , 48.7 ° C, 50.4 ° C, 52.4 ° C, 54.3 ° C, and 56.3 ° C in sequence for the sample 7 primer pair. Yes, Lanes 25 to 32 are the reaction results when the annealing temperature is 48.7 ° C, 50.4 ° C, 52.4 ° C , 54.3 ° C, 56.3 ° C, 58.6 ° C, 60 ° C, and 60.7 ° C in order for the sample 8 primer pair. .

  From the results of FIG. 5 and FIG. 6, the sample 1 primer pair confirmed the PCR amplification product up to ˜63.3 ° C., and the sample 2 primer pair confirmed the PCR amplification product only under the conditions of 61.4 ° C. to 63.3 ° C. Sample 3 primer pair, sample 5 primer pair and sample 6 primer pair cannot confirm PCR reaction product, sample 4 primer pair confirms PCR amplification product up to ~ 52.4 ° C, sample 7 primer pair The PCR amplification product was confirmed up to 47.3 ° C, and the sample 8 primer pair confirmed the PCR amplification product up to ~ 52.4 ° C.

  Based on the above results, PCR reaction is possible even with 1 base substitution at the 3 'end (sample 2), 2 base substitutions in the internal base sequence and addition of 5 bases complementary to the 3' end (sample 4) relative to the control group primer pair. At this time, it was confirmed that the reaction temperature of about 11 ° C. to about 10 ° C. was decreased as compared with the control primer pair. In addition, 1 base substitution of the internal base sequence and 3 base addition complementary to the 3 'end (sample 7) and 5 bases complementary to the 3 base end and 1 base replacement were added to the control primer pair (sample 7). In 8), a PCR reaction was possible, and in this case, it was confirmed that the reaction temperatures of about 16 ° C. and about 11 ° C. were reduced compared to the control primer pair, respectively.

Example 4. Melting curve verification experiment through dissociation step with respect to aprin primer production conditions A total of 8 types of aprin primer pairs confirmed in Example 3 were subjected to each using a melting curve method. Tm value was measured. Sample 1 forward primer in Example 3 (base sequence: 5'-CGT GTT TGT GCC TGT CCT GG-3 ', SEQ ID NO: 10) 0.3 nmole with 20 μl reaction standard per reaction and sample 2 forward primer (base Sequence: 5'-CGT GTT TGT GCC TGT CCT GN-3 ', SEQ ID NO: 12) 0.5 nmole and sample 3 forward primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GG-3', SEQ ID NO: 14 ) 1.0 nmole and sample 4 forward primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG AGA G-3 ', SEQ ID NO: 16) 0.5 nmole and sample 5 forward primer (base sequence: 5'- CGT GTT TNT GCN TGT NCT GGG AGA G-3 ', SEQ ID NO: 18) 1.0 nmole and Sample 6 forward primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG-3', SEQ ID NO: 20) 1.0 nmole Sample 7 forward primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG AG-3 ', SEQ ID NO: 22) 1.0 nmole, and sample 8 forward primer (base sequence: 5'-CGT GTT TGT GCN TGT CCT GGG AGA G-3 ', distribution To No. 24) 0.5nmole each common complementary reverse primer (nucleotide sequence: 5'-G GTA AGC AAG CAG GAC AAG AAG CGG-3 ', was used SEQ ID NO: 26) 0.3nmole. For other reaction additives, 10 μl reaction buffer was charged with ₂ μl of 20 μl of 100 mM Tris-HCl, 400 mM KCl, 50 mM MgCl ₂ , pH 9.0 per reaction, and 2 μl of 10 × SYBR Green. The SYBR Green is a fluorescent dye that intervenes in the minor groove region of the DNA double helix, and forms a double helix strongly at low temperatures. If the fluorescence value is high and the temperature is high, the double helix chain is loosened and becomes a single strand, so that the fluorescent dye cannot intervene and the fluorescence value cannot be detected. A melting curve was created using a real-time gene amplification device (Excycler ^™ , Bionicia), and the reaction conditions for this were as follows. After proceeding with a pre-denaturation step at 94 ° C. for 5 minutes, the temperature was maintained at 20 ° C. for 5 minutes, and after increasing for 1 second from 30 ° C. to 97 ° C. for 10 seconds, the fluorescence value was measured.

  FIG. 7 is a graph showing the melting curve results for the control group primer pair (sample 1) according to the replacement position of the aprin part, and line 1 is the melting curve created for the control group sample 1 primer pair. The line 2 is the melting curve value created for the sample 2 primer pair, and the line 3 is the melting curve value created for the sample 3 primer pair. FIG. 8 is a graph showing a melting curve result according to the number of substitutions of the internal base sequence with respect to the control group primer pair, and line 1 is a melting curve value created for the sample 1 primer pair as the control group. Line 2 is the melting curve value created for the sample 8 primer pair, line 3 is the melting curve value created for the sample 4 primer pair, and line 4 is created for the sample 5 primer pair. Melting curve value. FIG. 9 is a graph showing a melting curve result with the substitution of one base in the base group and the number of complementary bases added at the 3 ′ end with respect to the control group primer pair, and line 1 is Sample 1 which is a control group. Melting curve values created for the primer pair, line 2 is the melting curve value created for the sample 8 primer pair, and line 3 is the melting curve value created for the sample 7 primer pair. Yes, line 4 is the melting curve value created for the sample 6 primer pair, and line 5 is the melting curve value created for the sample 3 primer pair.

  From the above results, when a single base is replaced at the 3 ′ end with respect to the control primer pair, the Tm is reduced by about 1.5 ° C., and when a single base is replaced by the endogenous base sequence, the Tm is about 8 ° C. Decreased (see FIG. 7). Furthermore, when 5 complementary bases are added to the 3 ′ end, when the endogenous base sequence is replaced by 1, 2 or 3, the Tm is about 4 ° C. compared to the control primer pair, It was confirmed that the temperature decreased by about 10 ° C. and about 18 ° C. (see FIG. 8). In addition, when the base sequence was replaced by 1 base, when a complementary base was added to the 3 ′ end, it was confirmed that Tm increased by 0.5 ° C. every time 2 bases were added (FIG. 9). reference).

Example 5. Experiment for verifying the effectiveness of a PCR reaction through an basic primer in which an endogenous single nucleotide sequence showing polymorphism was replaced at the S gene site of hepatitis B virus (HBV)
An attempt was made to verify the effectiveness of the PCR reaction using the basic primer of the present invention, using an basic primer in which the endogenous single nucleotide sequence showing the polymorphism of the S gene site of hepatitis B virus was replaced, and a probe suitable for it. . In order to investigate the influence of the type of D-spacer, an basic primer having the same sequence was prepared using two types of D-spacers, that is, a D-spacer and a methoxy-D spacer (see FIG. 10). The base sequences of the prepared primers and probes are as shown in Table 2 below. The probes were used after labeling FAM fluorescent dye at the 5 ′ end and TAMRA fluorescent dye at the 3 ′ end.

The basic primers and probes were subjected to real-time PCR using an Applied Biosystems 7500 FAST real-time PCR system (Applied Biosystems, USA). Specifically, 20 μl reaction per reaction, forward primer (concentration 10 pmol) 1 μl, reverse primer (concentration 10 pmol) 1 μl, probe (concentration 5 pmol) 1 μl, 1 U Taq enzyme, other reaction additives Add 1 μl per 20 μl reaction of dNTP mixture (2.5 mM of each dATP, dCTP, dGTP and dTTP mixture), and add 10 mM reaction buffer for Taq enzyme with 200 mM Tris-HCl, 350 mM KCl, 15 mM MgCl ₂ , pH 9.0. 2 μl was added per 20 μl reaction. PCR reaction was performed under the following conditions. 1) 95 ° C for 12 minutes, 2) 95 ° C for 20 seconds, 3) 57 ° C for 40 seconds, 4) Repeat 40 times in steps 2) to 3). The results are shown in FIG. 11 and FIG.

  As shown in FIG. 11, when the primer was replaced with a deespacer in the one base and the primer replaced with a methoxydeespaceer, the same Ct value was shown under two conditions. Furthermore, as shown in FIG. 12, the same Ct value was shown when a primer with a normal base was used without replacing the endogenous 1 base at the same position as the primer with the intrinsic 1 base replaced with the methoxydi spacer. From the above results, it was confirmed that hepatitis B virus could be detected using the basic primer of the present invention.

SEQ ID NO: 1: p55 primer SEQ ID NO: 2: p53 primer SEQ ID NO: 3: No2-1F primer n is the basic part (dee spacer)
Sequence number 4: No2-1R primer n is an basic part (D spacer)
Sequence number 5: No2-2F primer n is an basic part (D spacer)
Sequence number 6: No2-2R primer n is an basic part (D spacer)
SEQ ID NO: 7: p63 primer SEQ ID NO: 8: No3-1R primer n is the basic part (D spacer)
Sequence number 9: No3-2R primer n is an basic part (D spacer)
SEQ ID NO: 10: F_AP_CONT primer SEQ ID NO: 11: R_AP_CONT primer SEQ ID NO: 12: F_AP_CONT_3-1N primer n is an basic part (dee spacer)
Sequence number 13: R_AP_CONT_3-1N primer n is an basic part (D spacer)
Sequence number 14: F_AP_CONT_I-1N primer n is an basic part (D spacer)
Sequence number 15: R_AP_CONT_I-1N primer n is an basic part (D spacer)
Sequence number 16: F_AP_CONT_I-2N_3 + 5M primer n is an basic part (D spacer)
Sequence number 17: R_AP_CONT_I-2N_3 + 5M primer n is an basic part (D spacer)
Sequence number 18: F_AP_CONT_I-3N_3 + 5M primer n is an basic part (dee spacer)
Sequence number 19: R_AP_CONT_I-3N_3 + 5M primer n is an basic part (D spacer)
Sequence number 20: F_AP_CONT_I-1N_3 + 1M primer n is an basic part (D spacer)
Sequence number 21: R_AP_CONT_1N_3 + 1M primer n is an basic part (D spacer)
Sequence number 22: F_AP_CONT_I-1N_3 + 3M primer n is an basic part (D spacer)
Sequence number 23: R_AP_CONT_I-1N_3 + 3M primer n is an abasic part (D spacer)
Sequence number 24: F_AP_CONT_I-1N_3 + 5M primer n is an basic part (D spacer)
Sequence number 25: R_AP_CONT_I-1N_3 + 5M primer n is an basic part (D spacer)
SEQ ID NO: 26: Common reverse primer SEQ ID NO: 27: Forward primer having a dee spacer n is an basic part (dee spacer)
SEQ ID NO: 28: forward primer with 1′-OMe-D spacer n is the basic part (1′-OMe-D spacer)
SEQ ID NO: 29: Forward primer having normal base SEQ ID NO: 30: Reverse primer having deespacer n is an basic part (deespacer)
SEQ ID NO: 31: reverse primer having 1′-OMe-D spacer n is an basic part (1′-OMe-D spacer)
SEQ ID NO: 32: Reverse primer having normal base SEQ ID NO: 33: Probe sequence

Claims (15)

  Primer for PCR amplification including the basic part.   The abasic part includes despacer (dSpacer, 5′-O-dimethoxytrityl-1 ′, 2′-dideoxyribose-3 ′-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), methoxydee Spacer (1'-OMe-dSpacer, 5'-O-dimethoxytrityl-1'-methoxy-2'-dideoxyribose-3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), PC spacer PC Spacer Phosphoramidite, [4- (4,4'-Dimethoxytrityloxy) butyramidomethyl) -1- (2-nitrophenyl) -ethyl] -2-cyanoethyl- (N, N-diisopropyl) -phosphoramidite) Ephosphoamidite (rSpacer CE Phosphoramidite, 5'-O-Dimethoxytrityl-1'-Deoxyribose-2'-O-Triisopropylsilyloxymethyl-3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), spacer Spacer C12 CE Phosphoramidite, 12- (4,4'-Dimethoxytrityloxy) -dodecyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), spacer phosphoramidite 18 (Spacer Phosphoramidite 18, 18-O-Dimethoxytritylhexae thyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite 9,9-O-Dimethoxytrityl-triethyleneglycol, 1-[(2-cyanoethyl)-( N, N-diisopropyl)]-phosphoramidite) and Spacer Phosphoramidite C3,3- (4,4'-Dimethoxytrityloxy) propyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)] The primer according to claim 1, wherein the primer is selected from the group consisting of -phosphoramidite).   The primer according to claim 1, wherein the number of basic portions in the primer ranges from 1 to 10.   The primer according to claim 3, wherein the number of basic portions in the primer is in the range of 1 to 3.   5. The primer according to any one of claims 1 to 4, wherein a base complementary to the template is added to the end of the primer in order to compensate for a decrease in Tm due to the basic portion.   The primer according to claim 1, wherein the basic portion is a portion corresponding to a mutated base site of a nucleic acid template to be hybridized.   The primer according to claim 1, wherein the basic part is a part corresponding to a polymorphic base site of a nucleic acid template to be hybridized.   8. A PCR amplification composition comprising a reaction buffer solution, four types of dNTPs, a DNA polymerase, and a PCR amplification primer according to any one of claims 1 to 7.   9. The composition of claim 8, wherein the composition further comprises a dye and / or a stabilizer.   10. The composition according to claim 9, wherein the dye is one or more selected from the group consisting of bromophenol blue, xylene cyanol, bromocresol red and cresol red.   10. The composition according to claim 9, wherein the stabilizer is at least one selected from the group consisting of gelatin, bovine serum albumin, Thesit, PEG-8000, and polyhydric alcohol.   8. A PCR amplification method comprising the steps of mixing a nucleic acid template with a PCR amplification composition comprising the PCR amplification primer according to any one of claims 1 to 7, and performing PCR amplification on the mixture.   13. The PCR amplification method according to claim 12, wherein the PCR amplification method is a method for commonly amplifying different nucleic acid templates having mutation sites in the base sequence.   13. The PCR amplification method according to claim 12, wherein the PCR amplification method is a method for commonly amplifying different nucleic acid templates having polymorphic sites in the base sequence.   One of the PCR amplification primer pairs is a PCR amplification primer according to any one of claims 1 to 7, and the other one is a normal primer that does not contain an basic part, The PCR amplification method according to claim 12.

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